1. Field of the Invention
The invention relates to novel lenti vector constructs designed to express recombinant full-length proteins or fragments thereof. The lenti constructs may be used for ex vivo or in vivo expression of a heterologous protein coding sequence by a cell or organ, or in vitro for the production of recombinant proteins by lenti vector-transduced cells.
2. Background of the Technology
Recombinant proteins as therapeutic modalities have found increasing use in recent years. Numerous recombinant protein-based therapies are in various stages of clinical development. One limitation to widespread clinical application of recombinant protein technology is the difficulty in production of proteins that include two or more coding sequences or domains such that the domains are expressed in the proper ratio with appropriate post-translational processing resulting in production of a functional heterodimeric molecule. A further limitation is the high cost associated with the ability to produced adequate levels of protein for clinical applications.
Monoclonal antibodies have been proven as effective therapeutics for cancer and other diseases. Current antibody therapy often involves repeat administration and long term treatment regimens, which are associated with a number of disadvantages, such as inconsistent serum levels, limited duration of efficacy per administration such that frequent readministration is required and high cost. The use of antibodies as diagnostic tools and therapeutic modalities has also found increasing use in recent years. One limitation to the widespread clinical application of antibody technology is that typically large amounts of antibody are required for therapeutic efficacy and the costs associated with production are significant. Chinese Hamster Ovarian (CHO) cells, SP20 and NSO2 myeloma cells are the most commonly used mammalian cell lines for commercial scale production of glycosylated human proteins such as antibodies. The yields obtained from mammalian cell line production typically range from 50-250 mg/L for 5-7 day culture in a batch fermentor or 300-1000 mg/L for 7-12 day cultures in fed batch fermentors. High-level production often relies upon gene amplification and selection of best performing clones that is time consuming and further increases the cost of development and production. In addition, stability issues with respect to antibody-producing cell lines are often evident following multiple passages.
Previous attempts to express full length recombinant proteins with two or more domains or chains (and thus two or more coding sequences or open reading frames (ORFs)) via recombinant DNA technology have met with limited success, typically resulting in unequal levels of expression of the two or more domains or chains of the protein or polypeptide and more importantly, a lower level of expression for the second coding sequence. In order to obtain optimal expression of a fully functional and biologically active protein or polypeptide that has two or more domains, substantially equimolar expression of the two or more domains is required. Conventional vectors that rely on dual promoter regulation of gene expression are invariably affected by promoter interaction (i.e., promoter interference) that may compromise equimolar or substantially equimolar expression of the genes.
Lentiviral vectors are a type of retroviral vector that can infect both dividing and non-dividing cells. They can be used to express protein from non-dividing or terminally differentiated cells such as neurons, macrophages, hematopoietic stem cells, retinal photoreceptors, muscle and liver cells, cell types for which other vector systems cannot be used effectively.
There remains a need for improved gene expression systems for production of recombinant proteins and polypeptides, in particular proteins and polypeptides that have two or more domains or chains, such that sufficient expression of a biologically active recombinant protein or polypeptide is achieved at commercially reasonable cost.
The present invention addresses this need by demonstrating the feasibility and use of lentivector constructs for the expression of functional recombinant proteins and polypeptides which have two or more domains.